Membrane lipoprotein with covalently linked lipids and fatty acids are ubiquitous components of biological membranes. In bacteria, lipid-modified proteins, which contain N-acyl glyceride-cysteine as the amino terminus, are synthesized as precursor proteins called prolipoproteins. Prolipoproteins undergo extensive posttranslational modification and processing reactions to form the mature lipoproteins. The biogenesis of lipoproteins in bacteria is intimately related to their secretion. Our long-term objectives are to study the biochemistry of lipoprotein maturation, to elucidate its mechanism of export and assembly into the bacterial cell envelope and to understand the regulation of these processes. We propose to continue our biochemical and genetic studies of lipoprotein biogenesis in both gram-negative and gram-positive bacteria. Our specific aims include (1) identification and characterization of prolipoprotein modification enzymes through enzyme purification, isolation of mutants defective in the activities of these enzyme purification, isolation of mutants defective in the activities of these enzymes, and cloning of the genes encoding these enzymes; (2) studies of the structures, membrane topology and assembly of prolipoprotein signal peptidase (signal peptidase II) in the cytoplasmic membrane of Escherichia coli; and (3) characterization of the x-ileS-lsp-orf149-orf316 operon in E. coli and other bacteria. Techniques to be employed include molecular genetics, recombinant DNA technology gene fusion, membrane biochemistry and bacterial physiological studies.